Kinetic studies on citrate synthase from pig heart

Johansson, Charlotta; Måhlén, Anders; Petterson, Gösta

doi:10.1016/0005-2744(73)90044-2

Cited by 25 publications

(6 citation statements)

References 14 publications

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“…Panel C of Figure 1 shows the spectrum of the binary complex of oxaloacetate with citrate synthase. No unbound OAA is present in these experiments (the Kdiss in 4 M urea is estimated to be 1-5 gM; Srere, 1966;Johansson et al, 1973). The maximum absorption occurs at 1697 ± 0.5 cm"1 with a line width of 10 ± 1 cm"1.…”

Section: Resultsmentioning

confidence: 75%

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Kurz¹,

Drysdale²

1987

Biochemistry

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The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.

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Section: Resultsmentioning

confidence: 75%

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Kurz¹,

Drysdale²

1987

Biochemistry

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“…Chemical Shifts of Oxaloacetate Bound to Citrate Synthase. Oxaloacetate binds unusually tightly to the enzyme in the binary complex (0.6 < Xdiss < 5 µ in 4 M urea ;Srere, 1966;Johansson et al, 1973). In the presence of a slight excess of enzyme (at an active site concentration of 1 mM), stable binary complexes can be studied by NMR techniques without possible ambiguities arising from the presence of unbound ligand.…”

Section: Resultsmentioning

confidence: 99%

Evidence from carbon-13 NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Kurz¹,

Ackerman²,

Drysdale³

1985

Biochemistry

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The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.

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“…A ternary-complex mechanism for an enzymic two-substrate reaction obeying a classical Dalzieltype rate equation can usually be shown to be either a rapidequilibrium or a compulsory-order type of mechanism [22]. According to Pettersson and co-workers [22][23][24] a discrimination between these two cases can be made from the fact that for a rapid-equilibrium mechanism Km,S, = Kd.sl and Kms2 = Kd,S2'…”

Section: Discussionmentioning

confidence: 99%

Ligand binding on to maize (Zea mays) malate synthase: a structural study

Beeckmans

Khan

Kanarek

et al. 1994

Biochemical Journal

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A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

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Kinetic studies on citrate synthase from pig heart

Cited by 25 publications

References 14 publications

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Evidence from carbon-13 NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Ligand binding on to maize (Zea mays) malate synthase: a structural study

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